Mtb 15 Project summary Over 300 known enzyme reactions depend on Coenzyme A (CoA), an essential co-factor formed from the adenylation of phosphopantetheine (Ppt). CoA sustains fatty acid synthesis and oxidation through its ability to carry and transfer acyl groups attached to the sulfhyryl on Ppt. Ppt's sulfhydryl also enables the synthesis of more complex lipids when Ppt is enzymatically transferred from CoA to apo-acyl carrier proteins (ACPs). Mycobacterium tuberculosis (Mtb) is now the leading cause of death from an infectious disease. In Mtb, the Ppt transferase encoded by rv2794c, called PptT, charges ACPs for the synthesis of key structural lipids?mycolic acids of the cell wall?and key virulence lipids?mycobactins that act as siderophores, and phthiocerol dimycoserosates (PDIMs) that suppress host immune reactions. PptT and the enzymes of CoA biosynthesis are attractive drug targets in Mtb, but success has been elusive. Enzymes have been cloned from several bacteria that can remove Ppt from holo-ACPs in vitro, but there has been no evidence for such a reaction in cells. Our recent discoveries alter this picture. We identified a drug-like compound that binds the active site of Mtb's PptT as confirmed by crystallography, only partially inhibits the recombinant enzyme and yet kills Mtb in vitro and in mice. The compound's mycobactericidal effect is ablated by mutations in or deletion of an adjacent gene of unknown function, rv2795c. We found that rv2795c encodes a phosphopantetheinyl hydrolase (PptH). This suggests that the PptT reaction is closely counter-balanced by the PptH reaction, so that partial inhibition of the forward reaction in the setting of the ongoing reverse reaction kills the cell. The goal of this application is to understand the physiologic functions of PptH and the conditions in which it becomes essential. We hypothesize that PptH contributes to a metabolic regulatory circuit in which Ppt is recycled into a CoA salvage path so that CoA can be rapidly re-deployed for reactions where it is most needed without de novo synthesis of the CoA precursor Ppt from ?-alanine and pantoate. Our first steps in testing this hypothesis are to define the functions (Aim 1), regulation (Aim 2), and interacting partners of PptH (Aim 3). PptH may then emerge as a novel drug target for future studies.